A great obstacle to HIV vaccine development is the resistance of primary isolates of HIV to neutralization. During the previous funding period for this grant, we have developed substantial data regarding mechanisms of neutralization resistance and the nature of primary virus cross-reactive neutralization. The aims of the current proposal build on our previous findings. Aim 1 of the proposed studies is to define structure-function relationships that determine the neutralization resistance (NR) and high infectivity (HI) phenotypes characteristics of the primary HIV-1-MN envelope, MNP. We have a family of envelopes that vary in neutralization resistance over a range of 205-fold. Among the parent clones and a large number of chimeric clones derived from them there is a very strong correlation between NR and infectivity. Our studies demonstrated that these phenotypes are determined by functional interactions of the leucine zipper (LZ) structure in gp41 with the amino and carboxyl terminal segments of gp120 and the downstream gp41 alpha helix. The mutations in gp120 that are present in the MNP clone cluster on the surface of the molecule in three regions: the CD4bs, the putative chemokine receptor binding site, and a region that we hypothesize to be the gp41 binding site. We hypothesize that a functional balance among the gp120 interactions with its three ligands and the interactions between the alpha helical regions in the gp41 ectodomain determine neutralization resistance. In this aim we will study molecular and functional mechanisms responsible for the global neutralization resistance, enhanced inefectivity phenotype. Aim 2 of the proposed studies is to define mechanisms that determine resistance or sensitivity of primary strains of HIV-1 to neutralization to antibodies directed against the conformation sensitive neutralization epitope at the apex of the V3 loop. Our previous studies have demonstrated that some primary isolates are neutralized by antibody against such epitope appears to contribute to primary virus cross-reactive neutralization. Since this is a highly conserved structure, it is a significant target for immunization. Studies will be conducted to determine mechanisms of resistance to neutralization at this epitope. Aim 3 of the proposed studies is to define characteristics that contribute to capacity of HIV envelopes to induce primary virus cross-reactive neutralizing antibodies. Previous studies have demonstrated that envelope with extensive neutralizing cross-reactivity from the donor of HIV- Neutralizing Serum (2) has an intermediate neutralization sensitivity phenotype and the capacity to induce primary virus cross reactive neutralization when administered in a alpha virus expression vector. Neutralization sensitivity and immunogenicity of envelopes from similar donors will be studied. These findings are highly relevant to vaccine development.